Lots of evidence suggests dipeptide repeat proteins translated from hexanucleotide repeat expansions in C9ORF72 interfere with transport of proteins in and out of the cell’s nucleus. However, an October 31 paper in Scientific Reports suggests that this effect must be indirect. Scientists led by Ludo Van Den Bosch, KU Leuven, Belgium, report that in several different cell models, the most toxic dipeptide repeat proteins (DPRs) leave nucleocytoplasmic transport intact. Only in a few cells that also contain stress granules do the researchers see any transport holdups, suggesting DPRs impair transport indirectly, possibly through a cellular-stress response. The results add one more piece to the puzzle of how these peptides cause disease.

  • Dipeptide repeat proteins leave nucleocytoplasmic transport unchanged.
  • Transport only slows down in cells that have stress granules.
  • The results suggest an indirect effect of DPRs on nuclear transport.

“The arginine-rich DPRs are key in the pathogenesis of C9ORF72 ALS/FTD,” wrote co-author Steven Boeynaems, Stanford University School of Medicine, California, to Alzforum. “Figuring out how they are toxic is important if we want to come up with novel therapeutic strategies.”

Hexanucleotide repeat expansions in C9ORF72 cause both amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The GGGGCC sequence in the first intron, repeated hundreds or thousands of times in patients, undergoes a noncanonical translation to yield five different DPRs—poly-GA (glycine-alanine), poly-GP (glycine-proline), poly-GR (glycine-arginine), poly-PA (proline-alanine), and poly-PR (proline-arginine). Some scientists believe they are toxic—particularly poly-GR and poly-PR—but don’t know how they interfere with normal cell function.

One body of research suggests that DPRs interfere with nucleocytoplasmic transport (Aug 2015 news; for a review, see Yuva-Aydemir et al., 2018). In fact, some studies report that poly-GR and poly-PR interact with nucleoporins that form the nucleopore complex through which molecules pass in and out of the nucleus. If so, then DPRs could directly interfere with nucleocytoplasmic transport (Lin et al., 2016). But is that the case?

To find out, first author Joni Vanneste and colleagues examined HeLa Kyoto cells that expressed a green reporter with a nuclear localization signal at one end and a nuclear export signal on the other. The reporter typically hangs out in the cytoplasm because the export signal is stronger. However, blocking export with leptomycin B causes the reporter to shuttle into, and accumulate in, the nucleus (see image below). In this way, the researchers measured import over time. When they added DPRs containing 20 repeats of either GR or PR to cultures of cells before and after treatment with leptomycin B, both nucleocytoplasmic export and import hummed along as normal, with no impairment.

Stuck Inside. HeLa Kyoto cells express a green reporter that passes in and out of the nucleus, but is usually cytoplasmic under control conditions (left). Adding leptomycin B (LMB) blocks export, shifting the reporter to the nucleus. Adding polyPR or polyGR dipeptides did not affect this movement. [Courtesy of Vanneste et al., 2019.]

Would a longer DPR—or perhaps one expressed inside the cell—make a difference? The authors expressed mCherry-labeled DPRs containing 100 repeats of either GA, PA, GR, or PR in HeLa cells. Once again, none affected nuclear export or import. However, the DPRs reduced the levels of protein translation, which suggested they were still toxic. The researchers found a similar result in iPSC-derived motor neurons from healthy donors and neuron-like SH-SY5Y cells. Only poly-GA caused a slight transport defect.

How do DPRs affect nucleocytoplasmic transport, if they don’t do so directly? The authors suggest they could trigger a cell-stress response in which post-translational modifications, relocalization, or degradation of transport factors could cause nuclear-transport problems. For instance, DPRs can induce stress granules, which could sequester proteins needed for nucleocytoplasmic transport. Interestingly, the group saw significant transport defects in the small percentage of HeLa Kyoto cells that contained stress granules.

Johnathan Cooper-Knock, University of Sheffield, England, U.K., was intrigued by that finding. “We know that stress-granule formation is a big part of motor-neuron disease,” he told Alzforum. “The way I interpret their result is that the PR and GR peptides don’t directly affect nucleocytoplasmic transport, but potentially have an indirect effect, which may be mediated via the formation of stress granules.”

Cooper-Knock cautioned that the study results overall were negative. While it is important to publish negative results, they can’t rule out a direct effect, he said. Even so, the finding could have therapeutic implications. “If you are going to try to rescue nucleocytoplasmic transport, this suggests you don’t want to target a direct interaction between PR and GR proteins with the nucleocytoplasmic pores,” he said.

Boeynaems agreed. The data instead suggest researchers could prevent nucleocytoplasmic-transport defects by targeting the cellular-stress response, for which drugs are available, he wrote to Alzforum.

Frank Hirth, King’s College London, called the results interesting but not surprising. He believes the nucleocytoplasmic-transport defect comes later in disease. “What may initiate disease is the accumulation of TDP-43 in the cytoplasm, which in turn can affect members of the nucleocytoplasmic-transport machinery.”

Hirth cautioned that some of the cells used in this study are not terminally differentiated, nondividing cells. He also cautioned that the results do not address the effect of the GGGGCC RNAs, which also form foci and could themselves interfere with and trap nucleoporins or other nucleocytoplasmic-transport components. Whether the expanded mRNA or the translated peptides are more toxic is a hot topic in ALS research.

The authors suggested that damaging nucleocytoplasmic transport requires more than just one component of C9ORF72-related dysfunction. For instance, it could be some combination of TDP-43 aggregates, RNA foci, cytoskeletal dysfunction, or aging. “It is good to tease apart different mechanisms individually,” agreed Brian Freibaum, St. Jude Children's Research Hospital, Memphis, Tennessee. “But some of the effects of C9ORF72 hexanucleotide repeat expansion likely come from the interplay of lots of factors—such as multiple DPRs, C9ORF72 RNA, and changes to protein expression—all happening at once.” Freibaum speculated that initially in a person’s life, there may be no direct interaction between GR or PR and the nuclear pore, but that may change with aging or neurodegeneration. “In the context of disease or stress, it’s possible that they then bind the nuclear pore and directly affect nucleocytoplasmic transport.”

The new findings contrast with a paper that reported 20-repeat poly-PR can block nucleocytoplasmic transport directly by binding proteins in the nuclear pore and restricting the movement of molecules in and out of the nucleus (Shi et al., 2017). The studies used different cell models, which could explain the discrepancy in findings, Vanneste and colleagues authors wrote. Other considerations are the concentration of LMB and the time allowed for the GFP reporter to enter the nucleus. Others pointed out that this reporter may diffuse into the nucleus, in which case any effect DRPs have on import/export machinery would be moot.—Gwyneth Dickey Zakaib


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News Citations

  1. ALS Gene Repeats Obstruct Traffic Between Nucleus and Cytoplasm

Paper Citations

  1. . Insights into C9ORF72-Related ALS/FTD from Drosophila and iPSC Models. Trends Neurosci. 2018 Jul;41(7):457-469. Epub 2018 May 2 PubMed.
  2. . Toxic PR Poly-Dipeptides Encoded by the C9orf72 Repeat Expansion Target LC Domain Polymers. Cell. 2016 Oct 20;167(3):789-802.e12. PubMed.
  3. . Toxic PRn poly-dipeptides encoded by the C9orf72 repeat expansion block nuclear import and export. Proc Natl Acad Sci U S A. 2017 Feb 14;114(7):E1111-E1117. Epub 2017 Jan 9 PubMed.

Further Reading


  1. . Nucleocytoplasmic transport defects in neurodegeneration - Cause or consequence?. Semin Cell Dev Biol. 2019 Jun 5; PubMed.

Primary Papers

  1. . C9orf72-generated poly-GR and poly-PR do not directly interfere with nucleocytoplasmic transport. Sci Rep. 2019 Oct 31;9(1):15728. PubMed.